Laccases (EC. 1.10.3.2 p-benzenediol: oxygen oxidoreductase) belong to a family of multi-copper oxidases. Laccases are widely distributed enzymes in higher plants, fungi, some insects and bacteria. They are characterized by low substrate specificity, oxidizing various substrates, including diphenols, polyphenols, different substituted phenols, diamines, aromatic amines, and even inorganic compounds like iodine. Laccases oxidize their substrates by a one-electron oxidation mechanism, and they use molecular oxygen as an electron acceptor. Among laccases the primary sequence, induction mechanism, physico-chemical (e.g. isoelectric point and carbohydrate content) and biochemical characteristics are variable. The copper binding sites of laccases are, however, strictly conserved.
Several laccase proteins and genes encoding these laccases have been previously isolated. WO 01/92498 describes a fungal laccase enzyme isolated from Melanocarpus albomyces strain. The enzyme having pH optimum within 5 to 8 and working at temperatures between 30 to 80° C. is well suited to industrial applications requiring high pH and temperature conditions whereas the majority of known fungal laccases function in an acidic pH range and are not very thermostable. The patent EP 0765394 B1 (corresponding U.S. Pat. No. 5,981,243) describes the cloning of a laccase gene from Myceliophthora thermophila and its expression in Aspergillus. The pH optimum of the laccase was 6.5 and the enzyme retained full activity 20 min in 60° C. U.S. Pat. No. 5,750,388 describes the cloning of a laccase gene from Scytalidium thermophilum and its expression in Aspergillus. The pH profiles of Scytalidium laccase activity has optimal pH of 7 and 4 for syringaldazine and ABTS oxidation, respectively. The laccase was more thermostable at neutral to alkaline pH than at acidic pH.
Laccases have many industrially potential applications, such as delignification of wood pulps, methods for treating lignin containing fibers, methods for treating wood fibers in order to functionalize them or glue the fibers, improval of the production of fuel ethanol from renewable raw materials, food applications (for example in baking or clarification of beer or wine), various bioremediative processes and textile applications, such as denim treatment, stain removal, treatment of various fibers for textile industry, methods for decolorizing dyes and methods for treating dye house effluents, or use in hair dyeing composition, in hard-surface cleaning or in detergent formulations.
“Stone washed” look or an abraded look has been denim producers' interest in recent years. Traditional stone washing with pumice stones reduces the strength of fabric and burdens the laundering apparatuses. Past years the trend has been towards enzymatic denim finishing processes. “Bleached look” of denim is normally obtained by means of sodium hypochlorite. So far this “chlorine bleaching” has been the most efficient bleaching method for denim dyed with Indigo, since almost all shades can be obtained. However, hypochlorite process is environmentally very harmful, it is difficult to control and it damages the fabric easily. It is also very inconvenient or even harmful method for the user, it cannot be used for Lycra containing products and antichlor treatment with several rinsing/washing steps is required. Intensive research has been underway for development of ecologically less harmful alternative for sodium hypochlorite, in particular laccases have been studied. So far the results with commercial laccase preparations cannot be compared to those obtained by “chlorine bleaching” as far as the effects and the looks are concerned. Heavy faded look has been very difficult or impossible to achieve without sodium hypochlorite.
WO 97/25468 describes the use of laccase in a method for providing to dyed denim an abraded look. The method comprises a cellulase treatment and simultaneous or subsequent treatment with a phenol oxidizing enzyme, such as laccase, and an enhancing agent, such as methylsyringate. Trametes villosa and Myceliophthora thermophila laccases are the examples of laccases in the patent publication.
Despite of the numerous publications describing laccases from various microorganisms, the prior art does not describe any laccase which could be used in denim treatment to replace the chemical bleaching agents. There is thus a need for novel laccase enzymes having more efficient oxidizing capacity, in particular more efficient oxidizing capacity of Indigo.
There is also a need for novel laccases, which would function more effectively and be more suitable for the various conditions in different applications.